Abstract 3567: Interaction of pralatrexate with the EGFR/MAPK pathway leads to synergistic activity with EGFR inhibitors

Abstract

Abstract Background: Pralatrexate (FOLOTYN®), an antifolate targeting dihydrofolate reductase, has high affinity for reduced folate carrier-1 and is an efficient substrate for polyglutamation by folypolyglutamyl synthetase. Combinations of cytotoxics and small molecule EGFR inhibitors (EGFRi's) have had little clinical success. Recent demonstration that intracellular folates may modulate survival signaling pathways, including EGFR/MAPK, suggests a potential for rational combinations of antifolates and EGFRi's. This study investigated potential roles of the EGFR/MAPK pathway in pralatrexate sensitivity and explored combinations of pralatrexate with EGFRi's. Materials and Methods: Anti-proliferative effects of pralatrexate were evaluated in a panel of human cancer cell lines. mRNA expression was analyzed by qRT-PCR and protein was evaluated by Western blot. Effects of drug combinations were evaluated using the Chou & Talalay method. Results: Pralatrexate displayed potent anti-proliferative effects in 9 of 15 human cancer cell lines, with HEP2 (head & neck [H&N]) and DU145 (prostate) cell lines showing IC50s of 50 nM and 15 nM, respectively. Sensitivity to pralatrexate correlated with higher pre-treatment mRNA expression of EGFR. After 5 hrs of exposure to 10 nM pralatrexate, increased phosphorylation of ERK1/2, MEK1/2, and the downstream target c-JUN was seen in HEP2 and DU145 cells. In addition, early activation of EGFR and EGFR protein degradation was observed. To further explore possible interactions between pralatrexate and the EGFR pathway, combination studies with EGFRi's were performed. Synergistic activity was observed in the MTT assay when pralatrexate was used in combination with erlotinib, gefitinib, and lapatinib in DU145, PC3, and LNCaP prostate and HEP2 H&N cancer cells. Synergistic activity was most pronounced when pralatrexate was given prior to the EGFRi's. Conclusions: Higher expression of EGFR was associated with pralatrexate sensitivity in a panel of human cancer cell lines. Pralatrexate treatment resulted in activation of the EGFR/MAPK pathway. Sequential administration of pralatrexate followed by EGFRi's resulted in synergistic anti-tumor activity. These findings provide rationale for clinical evaluation of pralatrexate in combination with EGFRi's, in particular in tumors sensitive to antifolates and EGFRi's such as NSCLC and H&N cancer. In addition, evaluation of pralatrexate in combination with inhibitors of downstream EGFR targets is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3567. doi:10.1158/1538-7445.AM2011-3567